Catecholamines are stored inside the chromaffin cell of the adrenal gland and inside nerve endings in storage granules at concentrations as high as 0.5 M. Studies using chromaffin granules isolated from bovine adrenal gland indicate that a carrier mediated active transport process is involved in transporting catecholamines from the cytosol to the inside of the granule utilizing the electrochemical energy produced by a separate Mg ATPase. The long range goal of this project is to determine the functional and structural characteristics of the catecholamine transporter. The objective of this proposal is to study the structural characteristics of transporter by purifying it to homogeneity, and determining its (a) reserpine binding and catecholamine transport characteristics, (b) the apparent molecular weight and number of subunits required for transport function, and (c) the amino acid composition and the presence of carbohydrates. Initial steps will be taken towards isolation of the mRNA for the transporter by developing polyclonal antibodies and isolation of the catecholamine transporter cDNA from a cDNA expression library. Purification of the transporter and isolation of its cDNA will provide us with tools which can be used to (a) obtain a better understanding of the one, two and three dimensional characteristics of the transporter, (b) the molecular mechanism involved in the transport process, (c) identify factors involved in regulation of synthesis of the transporter under normal and pathological conditions, (d) determine the difference between catecholamine serotonergic transporters in other areas of the nervous system, (e) identify other related proteins/genes or pseudogenes and (f) look for polymorphisms in the human population which can be correlated with disease states. Acquisition of polycolonal antibodies can be used (a) for isolation of the cDNA for the transporter, (b) for the identification of the transporter under different conditions, (c) as a probe to learn more about the functional characteristics of the transporter, (d) for the identification and/or purification of the catecholamine transporter from different tissues and (e) for determining the sidedness of storage granule ghosts. As part of the purification system we will develop an affinity chromatography system which can also be used for isolation and purification of chromaffin granules and catecholamine or serotonergic synaptic vesicles from the brain or peripheral nervous system.